In experiments proposed here we test the hypothesis that signaling through p21 activated kinase (Pak1) to protein phosphatase 2A (PP2A) is a novel mechanism of control of contractility by suppression of Ca-release units (CRU) in excitation contraction coupling (ECC) via effects on Ca2+ channel and ryanodine receptor function and stimulation of myofilament response to Ca2+ via sarcomeric protein dephosphorylation. Preliminary and published data in the current period of funding indicate that the function of Pak1 in integrated control of contractility involves signaling through 2-receptor/PKA phosphorylation of Pak1 and through sphingomyelin related lipid signaling that also activates Pak1. We also identified a novel mechanism of regulation of sarcomeric protein phosphorylation by various active forms of PKC6, which also acts in a signaling complex with Pak1. Aim #1 of our proposals is to test the hypothesis that the Pak1-PP2A signaling cascade is a novel mechanism of control of contractility acting by regulating the balance of CRU activity in ECC and myofilament response to Ca2+. Aim #2 is to determine the functional significance of diverse pathways of activation of PKC6 that induce dephosphorylation of cTnI and cTnT and phosphorylation of MyBP- C and Tm. Aim # 3 extends our studies on novel control of myofilament response to Ca2+ to our objective to determine if specific desensitization of the myofilaments to Ca2+ can serve as a therapeutic tool to prevent or attenuate the development of hypertrophy and dysfunction in transgenic mouse models of familial hypertrophic cardiomyopathy (HCM). Our preliminary and published data indicate that desensitization of myofilament response to calcium is able to rescue adverse effects in HCM-linked sarcomeric mutations in mouse models. Results of experiments proposed will provide insights into a previously unappreciated mode of activation of contractility, which provides new leads in translation medicine in cardiomyopathies. PUBLIC HEALTH RELEVANCE: Experiments proposed in the present application determine a new mechanism for control of the pressure developed in the heart that is responsible for ejection of blood. The new mechanism is likely to add to our understanding of heart failure, when pressure and ejection of blood is disturbed. Moreover, the new mechanism may lead to development of novel therapies for heart failure.